CD36-Mediated Hematoma Absorption
following Intracerebral Hemorrhage:
Negative Regulation by TLR4 Signaling
This information is current as
of June 16, 2017.
Huang Fang, Jing Chen, Sen Lin, PengFei Wang, YanChun
Wang, XiaoYi Xiong and QingWu Yang
J Immunol 2014; 192:5984-5992; Prepublished online 7 May
2014;
doi: 10.4049/jimmunol.1400054
http://www.jimmunol.org/content/192/12/5984
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Supplementary
Material
The Journal of Immunology
CD36-Mediated Hematoma Absorption following
Intracerebral Hemorrhage: Negative Regulation by TLR4
Signaling
Huang Fang,* Jing Chen,* Sen Lin,†,‡ PengFei Wang,* YanChun Wang,*
XiaoYi Xiong,* and QingWu Yang*
H
ematomas form when blood enters the cerebral parenchyma after an intracerebral hemorrhage (ICH); hematomas are the primary cause of neurologic deficits associated with ICH. The early stages of hematoma compression
cause deformation and mechanical injury to local brain tissues.
Secondary neuronal damage is caused by direct toxicity and inflammatory responses induced by the components and metabolic
products of late-stage hematomas and aggravates neurologic deficits (1). Therefore, effective hematoma removal is a goal of ICH
treatment, as hematoma removal can relieve mechanical compression, limit inflammatory injury, and promote the recovery of
neuronal function (2–4). Clinically, the rate of hematoma absorption in patients with ICH correlates with the degree of neu-
*Department of Neurology, Xinqiao Hospital, Third Military Medical University,
Chongqing 400037, China; †Department of Development and Regeneration Key
Laboratory of Sichuan Province, Chengdu Medical College, Chengdu 610083, China;
and ‡Department of Histoembryology and Neurobiology, Chengdu Medical College,
Chengdu 610083, China
Received for publication January 10, 2014. Accepted for publication April 5, 2014.
This work was supported by National Natural Science Foundation of China Grant
81271283 and National “973” Project Grant 2014CB541605.
Address correspondence and reprint requests to Prof. QingWu Yang, Department of
Neurology, Xinqiao Hospital, Third Military Medical University, 183 Xinqiao Main
Street, Shapingba District, Chongqing 400037, China. E-mail address: yangqwmlys@
hotmail.com
The online version of this article contains supplemental material.
Abbreviations used in this article: CT, computerized tomography; GFAP, glial fibrillary acidic protein; ICH, intracerebral hemorrhage; mRS, modified Rankin Scale;
NDS, neurological deficit score; NIHSS, National Institutes of Health Stroke Scale;
TAK-242, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1carboxylate; WT, wild-type.
This article is distributed under The American Association of Immunologists, Inc.,
Reuse Terms and Conditions for Author Choice articles.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400054
rologic deficit (5); however, the mechanism underlying hematoma
absorption remains unknown.
Scavenger receptors play important roles in the regulation of
phagocytosis in macrophages (6). CD36 is a type II scavenger
receptor that can bind to modified lipids, symmetric red cell
ghosts, or apoptotic neutrophils; this receptor plays an important
role in mediating phagocytosis (7, 8). Cells lacking phagocytic
abilities acquire phagocytic functions following transfection with
CD36 (9, 10). A peroxisome proliferator-activated receptor g agonist promotes hematoma absorption following ICH, which significantly reduces secondary inflammatory damages; the mechanism
may be associated with the upregulation of CD36 expression in the
microglia (2). However, the function of CD36 and the regulatory
mechanism underlying the effect of CD36 in hematoma absorption
are unclear.
TLRs are important components of the innate immune system
that recognize pathogen- and damaged-associated molecular patterns that could induce inflammatory responses (11). Many studies
have demonstrated that TLR4 participates in the development of
sterile inflammation and plays an important role in the inflammatory responses of the CNS, such as those that occur in the case
of cerebral ischemia and Parkinson’s disease (12). Our previous
studies showed that TLR4 activation following ICH results in
NF-kB activation via the MyD88 pathway, resulting in the production of large quantities of inflammatory factors, such as TNF-a
and IL-1b; these factors may cause inflammatory damage and aggravate neurologic deficits (13). Treatment with the TLR4 inhibitor
ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]-cyclohex-1-ene1-carboxylate (TAK-242; Takeda Pharmaceutical Company, Osaka,
Japan) decreases secondary inflammatory damage following ICH
(14). Other studies have shown that inflammatory factors such as
TNF-a, which is induced by the activation of TLR signaling pathways, downregulate the expression of CD36 in macrophages and
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Promoting hematoma absorption is a novel therapeutic strategy for intracerebral hemorrhage (ICH); however, the mechanism of
hematoma absorption is unclear. The present study explored the function and potential mechanism of CD36 in hematoma absorption using in vitro and in vivo ICH models. Hematoma absorption in CD36-deficient ICH patients was examined. Compared with
patients with normal CD36 expression, CD36-deficient ICH patients had slower hematoma adsorption and aggravated neurologic
deficits. CD36 expression in perihematomal tissues in wild-type mice following ICH was increased, whereas the hematoma absorption in CD362/2 mice was decreased. CD362/2 mice also showed aggravated neurologic deficits and increased TNF-a and IL-1b
expression levels. The phagocytic capacity of CD362/2 microglia for RBCs was also decreased. Additionally, the CD36 expression
in the perihematoma area after ICH in TLR42/2 and MyD882/2 mice was significantly increased, and hematoma absorption was
significantly promoted, which was significantly inhibited by an anti-CD36 Ab. In vitro, TNF-a and IL-1b significantly inhibited
the microglia expression of CD36 and reduced the microglia phagocytosis of RBCs. Finally, the TLR4 inhibitor TAK-242
upregulated CD36 expression in microglia, promoted hematoma absorption, increased catalase expression, and decreased the
H2O2 content. These results suggested that CD36 mediated hematoma absorption after ICH, and TLR4 signaling inhibited CD36
expression to slow hematoma absorption. TLR4 inhibition could promote hematoma absorption and significantly improve neurologic deficits following ICH. The Journal of Immunology, 2014, 192: 5984–5992.
The Journal of Immunology
affect cellular phagocytic functions that are essential to infectionrelated inflammatory responses (15). Additionally, CD36 expression
in perihematomal tissues is significantly upregulated in TLR42/2
mice subjected to ICH (16). Therefore, we hypothesized that
CD36 mediates hematoma absorption following ICH and that the
activation of TLR4 signaling following ICH produces inflammatory factors such as TNF-a that could downregulate CD36
expression and mediate hematoma absorption. To understand the
involvement of CD36 and TLR4 signaling in hematoma absorption following ICH, we modified CD36 expression in the relevant
cells. The goal of the present study was to further understand the
regulatory mechanism underlying hematoma absorption following ICH to identify treatment options that promote hematoma
absorption in patients with ICH.
Materials and Methods
5985
Collection of human brain tissue
Brain tissue samples from ICH patients were collected from the brain bank
of the Department of Neurosurgery of Xinqiao Hospital of the Third
Military Medical University (Chongqing, China). All autopsies had been
performed within 24 h of death. The inclusion criteria were as follows: 1) the
patient must not have had a previous ICH, and hemorrhage in the basal
ganglia was confirmed by CT scan; and 2) the patient had died within 48 h of
disease onset. The exclusion criteria were as follows: 1) the patient had
a traumatic hemorrhage or a hemorrhage caused by a coagulation dysfunction; 2) the patient had a history of acute or chronic infection or a known
inflammatory or autoimmune disease; and 3) the patient had a tumor,
cachexia, or had used immunosuppressive drugs for a prolonged period of
time. Three patients met the inclusion criteria and were enrolled. After the
human brain tissues were collected at autopsy, the perihematomal tissues
were used to detect CD36 expression using immunofluorescence. This
protocol for the clinical study was approved by the Ethics Committee of the
Xinqiao Hospital of the Third Military Medical University. All of the
subjects or authorized relatives signed informed consent forms. This study
did not have any medical ethics–related issues.
Clinical study
Animals
C57BL/6 mice (male, 8–10 wk old, 20–24 g) were obtained from the
Animal Center of the Third Military Medical University. TLR42/2,
MyD882/2, and CD362/2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All of the knockout mice had maintained the
C57BL/6 genetic background over the course of six generations of hybridization, and all were identified using appropriate methods. The mice
were housed in a clean environment and given ad libitum access to food
and water. The experimental protocol was approved by the Animal Management Committee of the Third Military Medical University. The mice
were randomly divided into groups, and the investigators were blinded to
the group allocation of the mice.
ICH model
The establishment of the ICH model has been described previously (14).
Briefly, the mice were anesthetized with 4% chloral hydrate and immobilized in a stereotactic apparatus (Stoelting, Wood Dale, IL). Twenty
microliters blood was collected from the tail vein, then directly injected
into the striatum without anticoagulant through stereotactic apparatus at
0.8 mm anterior and 2 mm lateral (on the left side) to the bregma and at
a depth of 3.5 mm. The blood was injected at a speed of 2 ml/min. The
needle was maintained in place for 10 min until the blood had coagulated;
the microinjector was then removed. The skull was sealed with bone wax,
and the scalp was sutured. The rectal temperature was maintained at ∼37˚C;
after recovery, the mice were given free access to water and food. In
parallel, sham mice received 20 ml striatal saline injection and were
subjected to the same manipulations as the ICH mice. The success rate of
the model was 90%; failed models and dead mice were excluded from
this study.
Analysis of the neurologic deficit score
The experiment was performed as described previously (14), and a 28-point
neurologic deficit scale was adopted. Circling behavior, climbing, front
limb symmetry, and body symmetry were assessed. The scoring was performed by two blinded laboratory investigators who were unaware of the
mouse groupings. The average score was the final score of each mouse.
Hematoma measurement
As in our previous report (25), the mice were anesthetized using a lethal
dose of chloral hydrate. After perfusion and fixation, the brains were removed and sectioned from the frontal lobe to the occipital lobe to prepare
coronal brain sections with a thickness of 1 mm. The fixed brain sections
were sequentially arranged, and Image-Pro Plus 5.0 image processing
software (Media Cybernetics, Bethesda, MD) was used to measure the ICH
volume (cubic millimeters). The hematoma volume was calculated using
the formula V = t 3 (A2 + … + An), where V is the hematoma volume, t is
the section thickness, and A is the bleeding area. Additionally, the hemoglobin content in the brain tissues was measured to further quantify
hematoma size (2). Blood (0, 2, 4, 8, 16, and 20 ml) was added to fresh
brain homogenates, and OD was then measured and recorded at 540 nm
using a spectrophotometer (Thermo Multiskan, Pittsburgh, PA). The obtained values were used to prepare the standard curve. The striatum was
removed after the ICH was initiated and dissolved in Drabkin’s reagent.
The supernatant of the homogenate was collected and measured using
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Certain people have CD36 expression deficiencies that affect the development of atherosclerosis (17, 18). To evaluate the effect of CD36 deficiency on hematoma absorption and neurologic deficits, we screened
patients with ICH for CD36 expression. Between July 2012 and June 2013,
209 patients with ICH were identified. The inclusion criteria were as
follows: 1) the patient was identified within 24 h of disease onset; 2) the
patient must not have had a previous ICH, and the hemorrhage in the basal
ganglia had to be confirmed by cranial computerized tomography (CT);
and 3) the hematoma must not have ruptured into the cerebral ventricles.
The exclusion criteria were as follows: 1) the patient was ,18 y or .80 y
old; 2) the patient chose to undergo surgical treatment; 3) the patient was
in a coma or died within 48 h of the ICH; 4) the ICH was caused by
a brain tumor, trauma, drug abuse, coagulation abnormalities, anticoagulation therapy, or vascular malformations; 5) the patient had had
an obvious inflammatory disease 6 mo prior to enrollment (e.g., acute
or chronic infectious diseases, systemic lupus erythematosus, rheumatism, or rheumatoid disease); 6) the patient had a nosocomial infection; 7) the patient had an acute myocardial infarction; 8) the patient
had acute or chronic liver damage; and 9) the patient was not compliant
with the study protocol or could not undergo all of the tests required by the
study. A total of 10 patients were excluded from the study, including 3
patients who died and 7 who were lost to follow-up. A total of 199 patients
met the inclusion criteria and were enrolled in the study. Upon admission to
the hospital, 10 ml blood from the median cubital vein was collected to
screen for CD36 deficiency. A cranial CT scan was performed on the day of
admission and 7 d after admission, as the hemorrhaged clot is generally
hyperdense relative to adjacent healthy cerebral parenchyma in the early
subacute phase of ICH (19, 20). The hematoma was visualized by CT
scanning (64-slice CT machine; Siemens, Munich, Germany). Hemorrhage
volumes were measured via computerized planimetry. The measurement of
hemorrhage volume by planimetrics is an established and accurate method
that uses computer-assisted image analysis (21). The volumes were expressed
in cubic centimeters. The volumes of hemorrhages within the ventricular
system were not measured. The following parameters were used for all CT
studies: section thickness, 5 mm; gap, 5 mm; pitch, 1; tube current, 304 mA;
and voltage, 120 kV. The hematoma absorption rate (percentage) was calculated with the following formula: [(hematoma volume at 7 d 2 hematoma
volume at admission)/hematoma volume at admission] 3 100. On admission
and 7, 14, and 30 d after admission, the patients were evaluated using
the National Institutes of Health Stroke Scale (NIHSS). Modified Rankin
Scale (mRS) scores were used to evaluate neurologic deficits in patients
3 mo after the onset of the ICH (22).
CD36 screening was performed using a sequence-specific primer PCR
assay. CD36 mutant primers were designed using information from previous
reports (23, 24). These mutations are the most common mutations that
cause CD36 expression defects in Asian populations (Supplemental
Table I). The patients lacking positive bands in the electrophoretogram
were considered CD36 deficient (Supplemental Fig. 1A). The mutations
were confirmed through sequencing analysis (data not shown). The types
of CD36 deficiency were identified through Western blot analysis of
monocytes (Supplemental Fig. 1B). Eleven patients with CD36 deficiency
in monocytes were identified (type I deficiency). Eleven patients with
normal CD36 expression had similar hematoma volumes at the same sites
and were selected to form the control group. Both groups of patients had
similar baseline data. The clinical data of the patients in these two groups
are shown in Supplemental Table II.
5986
a spectrophotometer; the hematoma volume (microliters) was then calculated using the standard curve.
Brain water content
As previously described (14), we randomly selected mice from each group
to measure the brain water content at 3 d after the model was successfully
constructed (n = 6). Briefly, the mice were anesthetized by i.p. injection,
and the cerebral tissues were removed. The samples were divided into five
parts: the ipsilateral cortex, the ipsilateral basal ganglia, the contralateral
cortex, the contralateral basal ganglia, and the cerebellum. The brain water
content (percentage) was calculated with the formula [(wet weight 2 dry
weight)/wet weight] 3 100.
Immunohistochemical staining
Real-time quantitative RT-PCR
Tissue and cellular RNA was extracted using the TRIzol reagent (Invitrogen,
Gaithersburg, MD), and cDNA was synthesized using the iScript cDNA
synthesis reagent (Bio-Rad, Hercules, CA) according to the manufacturers’
instructions. Real-time quantitative RT-PCR was performed in a 96-well plate
in the Bio-Rad iQ PCR machine (Bio-Rad, Hercules, CA) with the iQ SYBR
Green reagent. The primers were purchased from Shanghai Sangon Biotech.
The primer sequences used in the study are shown in Supplemental Table I.
The 22DDCT method was used to calculate relative gene expression levels (13).
Western blot
luted to a concentration of 108 cells/ml, added to the cultured microglia in
24-well plates at a ratio of 10:1, and cultured for 12 h. The microglia were
fixed and stained with rat anti-mouse CD11b (1:400; Millipore) followed
by Alexa Fluor 594 (1:500, donkey anti-rat; Invitrogen, Carlsbad, CA) and
Hoechst nuclear stain (Invitrogen, Eugene, OR). The observed results were
visualized using confocal microscopy and imaged. The intracellular localization of RBCs (green fluorescence) was validated by Z-stack fluorescence confocal imaging. Briefly, Z-stack images were collected as
a series of micrographs derived from different focal planes. Approximately
10 optical sections were captured from the top to the bottom of each
section in our study. The internalized RBCs were observed surrounded by
the red fluorescent cytomembrane of microglia when resectioning a
Z-stack was performed. Green fluorescent RBCs bound to the surface
were not surrounded by a red cytomembrane.
Flow cytometry
To detect the specific types of cells expressing CD36 in perihematomal
tissues, single-cell suspensions of perihematomal tissues were prepared as
described previously (16, 26). Briefly, after the mice were anesthetized with
chloral hydrate, cardiac perfusion was performed using cold sterile PBS; the
left brain was then removed immediately, placed in a C-tube (Miltenyi
Biotec, Bergisch Gladbach, Germany), mixed with lysis buffer, and ground
in a gentleMACS dissociator (Miltenyi Biotec). The brain tissue homogenate was filtered using a 40-mm sieve and incubated with demyelination
microbeads at 4˚C for 15 min. An L magnetic column was used to separate
the myelin sheath. The suspension that passed through the magnetic column
was used to generate brain tissue single-cell suspensions. To explore the
expression percentage of CD36 in each type of cell, the cells were stained
with the relevant surface markers (anti-mouse CD36-PerCP-eFluor 710
[1:200; eBioscience, San Diego, CA), anti-mouse CD11b Ab–PE [1:200;
Santa Cruz Biotechnology], and anti-mouse CD45 Ab–FITC (1:200;
eBioscience]) for 30 min and fixed with Fix buffer (BD Biosciences, San
Diego, CA) for 30 min at 4˚C. The cells were then incubated with BD
Cytoperm and buffer for 10 min at room temperature and refixed for
10 min. The cells were then incubated with anti-mouse GFAP–Fluor 660
(1:200; eBioscience) and anti-mouse neuron-specific b-III tubulin PerCP
(1:200; R&D Systems, Bingdon, U.K.) for 30 min at 4˚C. The data were
collected with FACSDiva 6.0 flow cytometer software (BD Biosciences)
and analyzed with FlowJo software (Tree Star, Ashland, OR). The microglia
were marked by CD45intCD11b+, whereas neurons were identified as b-III
tubulin+, and astrocytes were identified as GFAP+.
Analysis of phagocytosis of RBCs by microglia using flow
cytometry
As in our previous report (14), proteins from perihematomal tissues or
cultured microglia were resolved by SDS-PAGE and transferred onto
polyvinylidene fluoride membranes by electroblotting. The membranes
were incubated with mouse anti-human CD36 (for human blood, 1:400;
Wako Chemicals USA, Richmond, VA) or rabbit anti-mouse CD36 (for
perihematomal tissues and cells, 1:400; Abcam) at 4˚C overnight; GAPDH
(1:400; Santa Cruz Biotechnology, Dallas, TX) was used as a loading
control. The membranes were incubated with HRP-conjugated goat antimouse secondary Abs (1:2000) or HRP-conjugated goat anti-rabbit secondary Abs (1:2000; all from Sigma-Aldrich, St. Louis, MO) at 25˚C for
1.5 h. Bound Abs were visualized using a chemiluminescence detection
system. The signals were measured by scanning densitometry and
computer-assisted image analysis. Protein levels were calculated as the ratio
of the values corresponding to the band of the detected protein to the values
corresponding to the GAPDH band.
Following methods described in the literature (15), RBCs were stained with
CFSE and combined with the cultured microglia at a ratio of 10:1. After
coculturing for 12 h, the microglia were harvested and stained with anti–
CD11b Ab-PE for 30 min. The flow cytometry analysis showed that all of
the microglia were PE+. The microglia that had engulfed RBCs were PE+
CFSE+, and the nonengulfed RBCs in the supernatant were CFSE+. Three
indicators were used to assess the ability of microglia to phagocytize
RBCs: 1) the percentage of microglia that had engulfed RBCs (the total
number of PE+CFSE+ cells/all PE+ cells, expressed as percentage), 2) the
average CFSE fluorescence intensity of microglia that had engulfed RBCs
(PE+CFSE+), and 3) the number of nonengulfed RBCs (CFSE+) in the
supernatant. Additionally, changes in the microglia that had engulfed
RBCs were observed using the CD36 Ab (5 mg/ml; Abcam).
Microglial culture
To investigate the changes of CD36 expression in TLR42/2, MyD882/2
microglia following ICH, CFSE-stained RBCs were added to the different
microglial cultures at a concentration of 1 3 107 cells/ml and incubated for
12 h. To explore the different inflammatory factors on the influence of CD36
expression after ICH, CFSE-stained RBCs were added to the microglial
cultures and incubated for 12 h in the presence of the vehicle, TNF-a
(100 ng/ml; Life Technologies, Frederick, MD), IL-1b (100 ng/ml; R&D Systems, Minneapolis, MN), and IL-10 (10 ng/ml, BD Biosciences). The microglia
were collected and stained with anti-mouse CD36-PerCP-eFluor 710 and antimouse CD11b Ab–PE for 30 min; the expression of CD36 and changes in the
phagocytic ability of microglia were examined by flow cytometry.
Following methods described in the literature (2), the cells digested from
the cerebral hemispheres of postnatal day 1 newborn mice were cultured
for 2 wk. The loosely adherent microglia were harvested and inoculated
into six-well plates or on 12-mm coverslips in 24-well plates at a concentration of 1 3 105 cells/ml. The experiment was performed on the
following day. The cell purity was confirmed through immunohistochemistry using the microglia-specific rat anti-mouse CD11b Ab (1: 500;
Millipore). The purity of the microglia was .95%.
Phagocytosis in vitro
Following previously described methods (2), we added mouse RBCs to
cultures of microglia to model phagocytosis in vitro in a model of human
ICH. Mouse RBCs were purified via density gradient centrifugation and
labeled with CFSE (Invitrogen, Eugene, OR) for 30 min. RBCs were di-
Analysis of changes in microglial CD36 expression using flow
cytometry
TAK-242
TAK-242 (Takeda Pharmaceutical Company) is a TLR4-specific inhibitor
(14). CFSE-stained RBCs were mixed and cocultured with microglia at
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Following our previous methods (13), human and mouse perihematomal
tissues were fixed in 4% paraformaldehyde. The samples underwent gradient dehydration, embedding, freezing, and sectioning into 20-mm-thick
sections; CD36 expression was detected using double fluorescence immunohistochemistry. The following primary Abs were used in this experiment: mouse anti-human CD36 (1:200; LifeSpan, Seattle, WA), rabbit
anti-human Iba-1 (1:200; Wako Pure Chemical Industries, Osaka, Japan),
rabbit anti-human NeuN (1:200; Millipore, Darmstadt, Germany), rabbit
anti-human glial fibrillary acidic protein (GFAP; 1:100; Dako, Glostrup,
Denmark), rabbit anti-mouse CD36 (1:200; Abcam, Cambridge, U.K), rat
anti-mouse CD11b (1:200; Millipore), mouse anti-mouse GFAP (1:100;
Cell Signaling Technology, Danvers, MA), and mouse anti-mouse NeuN
(1:100; Millipore). The secondary Abs included Alexa Fluor 647 (1:200;
donkey anti-mouse), Alexa Fluor 594 (1:200; donkey anti-rabbit), and
Alexa Fluor 488 (1:200; donkey anti-rabbit, donkey anti-mouse, and
donkey anti-rat, all from Invitrogen, Carlsbad, CA). The observed results
were imaged by confocal microscopy (LSM 780, Carl Zeiss, Jena, Germany, and TCS Sp5, Leica, Mannheim, Germany).
CD36-MEDIATED HEMATOMA ABSORPTION
The Journal of Immunology
5987
a ratio of 10:1 in the presence of TAK-242 (1 mmol/l), TAK-242 (1 mmol/l)
plus TNF-a (100 ng/ml), TAK-242 (1 mmol/l) plus IL-1b (100 ng/ml), or
vehicle (solution for producing TAK-242) for 12 h. CD36 expression and
changes in phagocytosis in microglia were detected using flow cytometry.
To observe the effect of TAK-242 on hematoma absorption following ICH,
a previously described method was used (14); 6 h after the ICH model was
successfully established, the mice were i.p. injected with TAK-242 (3 mg/kg,
once per day for 5 d). The mice were divided into four groups: the sham
group, the ICH plus vehicle group (the solvent for TAK-242, administered
using the same volume as the TAK-242 group), the ICH plus TAK-242
group, and the ICH plus TAK-242 plus TNF-a group (first injecting TAK242 and then immediately injecting 3 mg TNF-a). The mice were sacrificed
5 d later, and the volume of the hematomas was measured.
Detection of catalase mRNA and H2O2 concentrations
Statistical analyses
All of the data are presented as the means 6 SD or the percentage. The analysis
was performed using SPSS 16.0 software. A repeated two-way ANOVA was
used to evaluate the differences in NIHSS scores for the clinical studies and in
the neurologic deficit score (NDS) for animal studies between the groups and
time points. The t test for independent samples or the Mann–Whitney U test
was used to compare two groups; comparisons among multiple groups were
examined using one-way ANOVA with a least significant difference post hoc
test. Differences were considered significant when p , 0.05.
FIGURE 1. Reduced hematoma absorption and aggravated neurologic
deficits in CD36-deficient patients with ICH. (A) Typical cranial CT
images of CD36-normal (n = 11) and CD36-deficient (n = 11) ICH
patients. Original magnification 310. (B) Comparison of hematoma volumes in the CD36-deficient and CD36-normal groups at admission and 7 d
after the onset of ICH. *p , 0.05 versus the CD36-normal group. (C)
Decreased absorption rate in the CD36-deficient patients relative to the
normal group. **p , 0.01 versus the CD36-normal group. (D) NIHSS
scores and (E) mRS scores for ICH patients. *p , 0.05 versus the CD36normal group, **p , 0.01 versus the CD36-normal group.
Results
CD36 affects hematoma absorption in ICH patients
The cranial CT images of patients in the CD36-deficient and CD36normal groups on the day of admission and 7 d after admission
showed that the hematoma volumes had changed (Fig. 1A). The
difference in the hematoma volumes of CD36-deficient and CD36normal patients was not significant at the onset of ICH; however,
the hematoma volumes of the patients in the CD36-deficient group
were larger than those of the patients in the CD36-normal group
at 7 d after the onset of ICH (Fig. 1B). The hematoma absorption
rate was lower in the CD36-deficient patients than in the CD36normal patients (Fig. 1C). The NIHSS scores of CD36-deficient
patients at 14 and 30 d were significantly higher than the scores of
the CD36-normal patients (Fig. 1D); the mRS scores of the deficient patients at 90 d were also significantly higher than those of
the normal patients (Fig. 1E). The results show that the hematoma
absorption rate in CD36-deficient patients was reduced and that
the neurologic deficits of these patients significantly aggravated
their condition. This finding suggests that CD36 plays an important role in promoting hematoma absorption in patients with ICH.
CD36 expression following ICH
Changes in CD36 expression in the perihematomal region were
observed using a mouse ICH model. At the corresponding time
points, perihematomal tissues were collected (the field selections
are shown in Supplemental Fig. 1C). The levels of CD36 mRNA
and protein in the perihematomal tissues were significantly higher
in the mice subjected to ICH than in the mice of the sham group.
The expression levels peaked at 3 d after the onset of the ICH and
then gradually decreased; on 7 d, the expression levels were still
higher in the treated mice than in the sham mouse group (Fig. 2A,
2B). No time-dependent alterations in CD36 expression were
observed in the sham group mice (data not shown). The results of
the flow cytometry analysis of cells from the perihematomal tissues showed that the expression levels of CD36 in neurons,
astrocytes, and microglia were significantly higher in the experimental mice at 3 d after the onset of the ICH than in the sham
group; however, microglia became CD36+ at a significantly more
rapid rate than did neurons and astrocytes (Fig. 2C). The immunofluorescent staining of perihematomal tissues from ICH patients
produced results that were consistent with the results obtained
with flow cytometry (Fig. 2D). Similarly, the results of immunofluorescent staining of the perihematoma of mice at 3 d after ICH
were consistent with the patients who had experienced ICH
(Supplemental Fig. 1D). As the resident phagocytes of the brain,
microglia were involved in regulating the inflammatory response
that occurs following ICH. Our results showed that CD36 was
abundantly and mainly expressed in the microglia surrounding the
perihematomal tissues following ICH, suggesting that CD36 plays
a role in the pathological processes associated with ICH.
CD36-mediated hematoma absorption following ICH
The ICH model was used to evaluate the role of CD36 in the
process of hematoma absorption. The absorption and the volume of
hematomas were lower and larger, respectively, in CD362/2 mice
than in wild-type (WT) mice (Fig. 3A, 3B). Additionally, the NDS
and the brain water content of CD362/2 mice were significantly
higher than those of the mice in the sham group (Fig. 3C, 3D); the
mRNA levels of TNF-a and IL-1b were also higher in the perihematomal tissues of the CD362/2 mice than in the mice of the
sham group. In contrast, IL-10 expression levels were significantly
lower in the deficient mice than in the mice of the sham group
(Fig. 3E).
To further confirm the involvement of CD36 in the promotion
of hematoma absorption, we observed the phagocytosis of RBCs
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Following previously described methods (2), the microglial suspensions
were divided into two groups: the vehicle group and the TAK-242 group.
RBCs were added at a ratio of 10:1 in the presence of vehicle or TAK-242
(1 mmol/l). After culturing for 12 h, the microglia were collected and the
changes in catalase mRNA expression were detected using quantitative
PCR. The catalase primer was purchased from Shanghai Sangon Biotech,
and the primer detail information is shown in Supplemental Table I. Additionally, the culture medium was collected and H2O2 concentrations were
measured with a detection kit (Invitrogen) according to the manufacturer’s
instructions.
5988
by different microglia in a simulated in vitro ICH model (2).
Representative images showing the number of RBCs engulfed by
microglia showed that significantly fewer RBCs were engulfed in
the CD362/2 group than in the WT group. The ability to phagocytize RBCs was decreased in WT microglia treated with a CD36
Ab, whereas no change in the phagocytic ability of WT microglia
treated with IgG was observed (Fig. 4A). Flow cytometry was
conducted to analyze the ability of microglia to phagocytize
RBCs; the CD362/2 group had significantly fewer PE+CFSE+
microglia that engulfed RBCs than did the WT group (Fig. 4B).
Additionally, the average fluorescence intensity of CFSE in the
PE+CFSE+ microglia was significantly decreased in the CD362/2
group (Fig. 4C). Accordingly, the number of nonengulfed RBCs in
the supernatant was higher in the CD362/2 group than in the WT
group (Fig. 4D). Similar results were observed when the microglia
were treated with anti-CD36 Abs; in contrast, the phagocytosis of
RBCs by microglia in the WT group was not significantly affected
by treatment with IgG (Fig. 4B–D). These results suggest that
CD36 plays an important role in promoting hematoma absorption.
TLR4/MyD88 signaling inhibits CD36 expression and
decreases hematoma absorption
The effect of TLR4 signaling on CD36 expression and hematoma
absorption was explored using in vitro and in vivo ICH models.
FIGURE 3. CD36-mediated hematoma absorption in mice with ICH.
(A) Serial coronal sections of mouse brain tissues 5 d after the onset of
ICH. Original magnification 31.5. (B) Comparison of hematoma volumes
between WT and CD362/2 mice. The data in the left and right panels are
the Image-Pro Plus and hemoglobin detection results, respectively. **p ,
0.01 versus the WT group, n = 6. (C) The NDS of WT and CD362/2 mice
at 1, 3, 5, and 7 d after the onset of ICH. *p , 0.05 versus the WT group at
the corresponding time points, n = 6. (D) Brain water content and (E)
mRNA expression of inflammatory factors 3 d after the onset of ICH. The
data in (E) were assessed by real-time quantitative RT-PCR and were
expressed as fold increases relative to naive animals. *p , 0.05 versus the
WT group; **p , 0.01 versus the WT group, n = 6. Cereb, cerebellum;
Cont BG, contralateral basal ganglia; Cont CX, contralateral cortex; Ipsi
BG, ipsilateral basal ganglia; Ipsi CX, ipsilateral cortex.
CD36 expression in the perihematoma of the MyD882/2 and
TLR42/2 mice was significantly increased at 3 d after the onset of
ICH, as observed in the Western blot (Fig. 5A), and in the in vitro
ICH model significantly increased CD36 expression was also
detected in the MyD882/2 and TLR42/2 microglia by flow cytometry (Fig. 5B). Moreover, the significantly decreased hematoma volumes were found in the MyD882/2 and TLR42/2 mice
(Fig. 5C), and flow cytometry showed that the MyD882/2 and
TLR42/2 groups had more PE+CFSE+ microglia than did the WT
group in vitro (Fig. 5D). The average CFSE fluorescence intensity
was higher in PE+CFSE+ microglia from the MyD882/2 and
TLR42/2 groups than in the microglia of the WT group (Fig. 5E),
and significantly fewer RBCs were nonphagocytosed in the
MyD882/2 and TLR42/2 groups (Fig. 5F). The phagocytic
capacity of MyD882/2 and TLR42/2 microglia treated with antiCD36 Abs was significantly inhibited; in contrast, the phagocytic
capacity of MyD882/2 and TLR42/2 microglia treated with IgG
was not affected (Fig. 5D–F). These results indicate that TLR4
signaling may downregulate CD36 expression and inhibit hematoma absorption.
The effect of inflammatory molecules released downstream of
TLR4 signaling (such as TNF-a) on the phagocytic capacity of
microglia was investigated. Western blot analysis showed that the
addition of RBCs to microglial cultures in the presence of TNF-a
and IL-1b significantly decreased CD36 expression; in contrast,
the addition of IL-10 significantly upregulated CD36 expression
(Fig. 6A). The microglia CD36 expression levels observed using
flow cytometry were consistent with the above results (Fig. 6B).
The percentage of PE+CFSE+ microglia in the TNF-a– and IL1b–treated groups was lower than in the vehicle-treated group
(Fig. 6C); additionally, the average fluorescence intensity of CFSE
in PE+CFSE+ microglia was decreased (Fig. 6D) and the number
of nonphagocytosed RBCs was increased (Fig. 7E) in the treated
groups. In contrast, in the IL-10–treated group, the percentage of
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FIGURE 2. Enhanced CD36 expression in perihematomas following
ICH. (A) Detection of CD36 mRNA expression in mice subjected to ICH
using real-time quantitative RT-PCR. The data are expressed as fold
increases relative to naive animals. **p , 0.01 versus sham, n = 6. (B)
Detection of CD36 protein expression in mice subjected to ICH using
Western blot. **p , 0.01 versus sham, n = 6. (C) Detection of CD36
expression in astrocytes, neurons, and microglia in the perihematomal
tissues of mice using flow cytometry at 3 d after ICH. The left panel is the
representative flow cytometry plot and the lower right panel is the percentage of CD36+ cells in neurons, astrocytes, and microglia. Microglia
were marked by CD45intCD11b+, whereas neurons were identified as b-III
tubulin+, and astrocytes were identified as GFAP+. **p , 0.01 versus
sham, ##p , 0.01 versus astrocytes or neurons, n = 3. (D) Detection of
CD36 expression in human perihematomal tissues of ICH patients using
fluorescence immunohistochemistry; CD36 expression was labeled with
a CD36 Ab (red), astrocytes, neurons, and microglia were labeled with
GFAP (green), Neun (green), and Iba-1 (green), respectively. The merged
images of the overlay of CD36 together with astrocytes, neurons, and
microglia were shown as yellow, and the nuclei were stained with DAPI (blue).
The arrows indicate positive cells. Scale bars, 80 mm.
CD36-MEDIATED HEMATOMA ABSORPTION
The Journal of Immunology
5989
microglia and promote RBC phagocytosis. In the TAK-242 plus
TNF-a group and the TAK-242 plus IL-1b group, the percentage
of PE+CFSE+ microglia and average fluorescence intensity of
CFSE were reduced; however, the number of RBCs remaining in
the supernatant was increased (Fig. 7C–E), and the effect of
TNF-a was more apparent. These results suggest that the TLR4
signaling pathway regulates CD36 expression and its function
via inflammatory factors, especially TNF-a. In the in vitro ICH
model, the expression of catalase mRNA was significantly increased (Fig. 7F), and the H2O2 content was significantly decreased (Fig. 7G) in the microglia of the TAK-242 group. Finally,
when TAK-242 was applied 6 h after the onset of the in vivo ICH
models, the hematoma volume in treated mice was significantly
lower than in mice of the vehicle control group 5 d after the onset
of the hematoma. Because TNF-a had an inhibiting effect of
CD36 expression in vitro, after the first injection of TAK-242, we
immediately injected TNF-a, which showed that the protective
effect could be weakened by TNF-a (Fig. 7H).
FIGURE 4. CD36-mediated engulfment of RBCs by microglia. (A)
Observation of the phagocytosis of RBCs (CFSE-labeled, green) by
microglia (Alexa Fluor 594–labeled, red) using confocal microscopy. The
nuclei were stained with Hoechst. Serial sections along the z-axis were
acquired and compiled as images. The x–z-axis is below, and the y–z-axis is
on the right. The arrows indicate the RBCs engulfed by microglia. Scale bars,
40 mm. Detection of changes in the phagocytic ability of microglia using
flow cytometry is shown. Microglia were stained with anti-mouse CD11b-PE
and RBCs were stained with CFSE. (B) Representative flow cytometry plot
(left panel) and the percentage of PE+CFSE+ microglia that had engulfed
RBCs (right panel). (C) Average fluorescence intensity of CFSE in the PE+
CFSE+ microglia. (D) The number of nonphagocytosed RBCs in the supernatant. **p , 0.01 versus the WT microglia group, n = 6 for all graphs.
PE+CFSE+ microglia and the average fluorescence intensity of
CFSE in PE+CFSE+ microglia increased and the number of nonphagocytosed RBCs decreased (Fig. 6C–E). These results suggest
that molecules released downstream of TLR4 signaling (such as
TNF-a and IL-1b) can inhibit hematoma absorption following
ICH by downregulating CD36 expression.
TAK-242 increases CD36 expression, promotes hematoma
absorption, and increases catalase expression
Our previous studies showed that TAK-242 could reduce secondary
injury caused by ICH by inhibiting TLR4 signaling (14). To observe the effect of TAK-242 on hematoma absorption, CD36 expression levels were measured in microglia cocultured with RBCs
in the presence of TAK-242, TAK-242 plus TNF-a, TAK-242 plus
IL-1b, or vehicle (solution for TAK-242) for 12 h. Western blot
analysis showed that CD36 expression levels were higher in the
TAK-242 group than in the vehicle group, whereas the TAK-242–
promoted expression of CD36 was weakened by TNF-a and IL1b; TNF-a had an apparent effect in this process (Fig. 7A).
However, when the microglia were treated with TAK-242 only,
CD36 expression levels did not change significantly (data not
shown). The CD36 expression changes determined by flow
cytometry were consistent with the Western blot results (Fig. 7B).
Flow cytometry showed that the percentage of PE+CFSE+
microglia in the TAK-242 group was increased (Fig. 7C); additionally, the average fluorescence intensity of CFSE in PE+CFSE+
microglia was increased (Fig. 7D), and the number of RBCs
remaining in the supernatant was decreased (Fig. 7E). These
findings suggest that TAK-242 can upregulate CD36 expression in
Clinical trials evaluating the surgical removal of hematomas have
not achieved the expected results (27). The promotion of hematoma absorption in cerebral tissues represents a new potential
treatment option for patients with ICH (2, 3). However, the regulatory mechanism underlying hematoma absorption is still unclear. The present study showed that CD36-mediated hematoma
absorption occurs in ICH patients and that this absorption is significantly associated with patient prognosis. The results of in vitro
and in vivo experiments related to ICH further confirmed that
CD36 promotes hematoma absorption. After the onset of ICH,
inflammatory factors induced by the activation of the TLR4 signaling pathway downregulate CD36 expression and inhibit hematoma absorption. The application of TLR4 inhibitors could
upregulate CD36 expression and increase hematoma absorption.
To our knowledge, our study is the first to clarify the mechanism
underlying hematoma absorption in patients with ICH; the results
of this study provide reliable experimental data that may contribute to the development of clinical treatments that promote
hematoma absorption in patients with ICH.
Two types of CD36 expression deficiency exist: patients with
a type I deficiency have deficient CD36 expression in monocytes
and platelets, and patients with a type II deficiency have deficient
expression in platelets only (28). The cells from patients with
a type I CD36 deficiency have a reduced ability to phagocytize
oxidatively modified low-density lipoprotein; these patients often
experience complications such as insulin resistance, hypertension,
and the aggravation of atherosclerosis. Additionally, the incidence
of coronary artery disease is three times higher in patients with
a type I CD36 deficiency than in normal individuals (17). CD36
expression deficiencies are rarely encountered among European
and American whites; however, .2% of Asians and Africans are
afflicted with a CD36 deficiency, and the prevalence of this deficiency in Japan ranges from 3 to 11% (29, 30). We used a sequence-specific primer PCR assay to detect CD36 expression
deficiencies in ICH patients. Sequencing and Western blot analyses identified 11 patients with a type I deficiency from among
199 patients (5.5%). The high prevalence of this deficiency in our
patient population may be due to regional differences, small
sample sizes, and the fact that only patients with ICH were selected. We found that type I CD36–deficient ICH patients had
hematomas that were less well absorbed, and they had significantly more severe neurologic deficits than did patients with
normal CD36 expression. These results suggest that CD36 pro-
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Discussion
5990
CD36-MEDIATED HEMATOMA ABSORPTION
motes hematoma absorption after ICH and confirm that the rate at
which the hematoma is absorbed positively correlated with patient
prognosis. The destruction of the blood–brain barrier following
ICH can allow peripheral mononuclear macrophages to enter
perihematomal tissues (16); therefore, it has been suggested that
the CD36+ peripheral mononuclear macrophages that enter into
the perihematomal tissues participate in hematoma absorption.
Microglia are resident brain mononuclear macrophages; our data
showed that CD36 was mainly expressed in microglia near the
perihematomal tissues following ICH and that microglial CD36
played an important role in enabling hematoma absorption. These
two types of cells are thought to play important roles in hematoma
absorption; however, further studies are required to determine
which cell type is more important. Additionally, because our study
was conducted at a single center with a small sample size, our
conclusions must be confirmed in multicenter studies with larger
sample sizes. The relevant studies are currently in progress.
We found that CD36 significantly promotes hematoma absorption following ICH; however, the regulatory mechanism underlying this process remains unclear. Studies have shown that
inflammatory factors such as TNF-a that are induced by TLR
signaling can regulate the expression of CD36 in macrophages
that are very important in infection-related inflammatory responses
(15). This suggests that the activation of TLR4 signaling following
ICH may have a negative regulatory effect on CD36 expression. Our
study showed three significant results. First, CD36 expression was
significantly upregulated in perihematomal tissues in TLR42/2 and
MyD882/2 mice. Second, hematoma absorption was significantly
increased in TLR42/2 and MyD882/2 mice. The microglia of
TLR42/2 and MyD882/2 mice engulfed more RBCs than did
WT microglia; however, after treatment with anti-CD36 Abs, the
phagocytic ability of TLR42/2 and MyD882/2 microglia was
significantly decreased, suggesting that TLR4 signaling regulated
hematoma absorption through CD36. Third, the inflammatory
factors TNF-a and IL-1b that were induced by the activation of
TLR4 signaling downregulated microglial CD36 expression and
inhibited hematoma absorption. These results suggest that CD36
mediates hematoma absorption; however, the activation of TLR4
signaling following ICH can activate NF-kB via the MyD88
pathway, resulting in the production of large amounts of TNF-a
and IL-1b (13). TNF-a and IL-1b can inhibit CD36 expression and
slow hematoma absorption. Sansing et al. (16) reported that CD36
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FIGURE 5. TLR4 signaling downregulates CD36
expression and increases hematoma absorption. (A)
Detection of CD36 expression in perihematomal tissues
3 d after the onset of ICH using Western blot. **p ,
0.01 versus the WT group, n = 6. (B) Detection
of microglia CD36 expression using flow cytometry.
**p , 0.01 versus the WT group, n = 6. (C) Five days
after the onset of ICH, the hematoma volume was
smaller in MyD882/2 and TLR42/2 mice than in WT
mice. Measurement of hematoma volume using ImagePro Plus (left panel) and hemoglobin detection (right
panel) are shown. **p , 0.01 versus the WT group, n =
6. Detection of changes in the phagocytic capacity of
microglia using flow cytometry is depicted. Microglia
were stained with anti-mouse CD11b-PE, and RBCs
were stained with CFSE. (D) Representative flow
cytometry plot (left panel) and the percentage of PE+
CFSE+ microglia that had engulfed RBCs (right panel);
(E) average fluorescence intensity of CFSE in PE+
CFSE+ microglia; (F) number of nonphagocytosed
RBCs in the supernatant. **p , 0.01 versus the WT
microglia group, ##p , 0.01 versus the groups with
anti-CD36 Abs, n = 6 for all flow cytometry graphs.
The Journal of Immunology
expression was upregulated in the TLR4 knockout mice following ICH, suggesting that TLR4 may have participated in the
CD36 regulation, which was consistent with our results that
TLR4 regulated the CD36 expression in the ICH. However, recent
research suggests that under the stimulation of oxidized lowdensity lipoprotein and amyloid-b, CD36 functions as a TLR4/
TLR6 coreceptor and participated in the early inflammatory
mediators expression in atherosclerosis and Alzheimer disease
(31). The reasons for differing results may be related to using
different models, and CD36 may have different functions varying
among diseases. Moreover, our results suggest that IL-10 can
promote CD36 expression and enhance the ability of microglia to
phagocytize RBCs; however, IL-10 levels were not obviously
increased 1 and 3 d after ICH (32), suggesting that the protective
role of IL-10 during the acute phase of cerebral hemorrhage was
limited. Therefore, residual blood components activated TLR4
signaling and produced inflammatory injury, thus initiating an
inflammatory cascade reaction and aggravating neurologic deficits. The inhibition of the activation of TLR4 signaling could
therefore inhibit inflammatory responses and promote hematoma
absorption.
FIGURE 7. TAK-242 (TAK) increases CD36 expression and promotes
hematoma absorption. (A) Detection of CD36 protein expression using
Western blot. **p , 0.01 versus the vehicle group, #p , 0.05 versus the
TAK group, ##p , 0.01 versus the TAK group, n = 6. (B) Detection of
microglia CD36 expression using flow cytometry. **p , 0.01 versus the
vehicle group, ##p , 0.01 versus the TAK group, n = 6. Detection of
changes in the phagocytic capacity of microglia using flow cytometry.
Microglia were stained with anti-mouse CD11b-PE, and RBCs were
stained with CFSE. (C) A representative flow cytometry plot (left panel)
displaying the percentage of PE+CFSE+ microglia (right panel) that had
engulfed RBCs. **p , 0.01 versus the vehicle group, ##p , 0.01 versus
the TAK group, n = 6. (D) The average fluorescence intensity of CFSE in
PE+CFSE+ microglia. **p , 0.01 versus the vehicle group, ##p , 0.01
versus the TAK group, n = 6. (E) The number of nonphagocytosed RBCs in
the supernatant. **p , 0.01 versus the vehicle group, ##p , 0.01 versus the
TAK group, n = 6. (F) Detection of microglial catalase expression using
real-time quantitative RT-PCR. The data are expressed as fold increases
relative to the vehicle group. **p , 0.01, n = 6. (G) The changes in H2O2
content in the supernatant were measured. The results are presented as fold
changes relative to vehicle group. **p , 0.01, n = 6. (H) Changes in
hematoma volume in mice at 5 d after the onset of ICH. Measurement of
hematoma volume using Image-Pro Plus (left panel) and detection of
hemoglobin (right panel). **p , 0.01 versus the ICH plus vehicle group,
#
p , 0.05 versus the ICH plus TAK plus TNF-a group, n = 6.
Our recent studies have shown that the TLR4 inhibitor TAK-242
significantly relieves inflammatory injury after ICH and improves
neurologic deficits (14). The present study further showed that
TAK-242 upregulated CD36 expression in microglia, increasing
the phagocytic capacity of microglia and improving hematoma
absorption in ICH mice. However, simultaneous use of TNF-a and
IL-1b could weaken the ability of TAK-242 to upregulate CD36
expression. This finding suggests that the TAK-242–mediated
mechanism underlying the relief of inflammatory injury in mice
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FIGURE 6. Effects of TNF-a, IL-1b, and IL-10 on CD36 expression
and phagocytosis in microglia. (A) Detection of changes in microglial
CD36 protein levels using Western blot. *p , 0.05 versus the vehicle
group, **p , 0.01 versus the vehicle group, n = 6. (B) Detection of
microglial CD36 expression using flow cytometry. **p , 0.01 versus the
vehicle group, n = 6. Detection of changes in the phagocytic ability of
microglia using flow cytometry. Microglia were stained with anti-mouse
CD11b-PE, and RBCs were stained with CFSE. (C) Representative flow
cytometry plot (left panel) and the percentage of PE+CFSE+ microglia that
had engulfed RBCs (right panel). **p , 0.01 versus the vehicle group,
n = 6. (D) Average fluorescence intensity of CFSE in PE+CFSE+
microglia. **p , 0.01 versus the vehicle group, n = 6. (E) Number of
nonphagocytosed RBCs in the supernatant. *p , 0.05 versus the vehicle
group, **p , 0.01 versus the vehicle group, n = 6.
5991
5992
with ICH was associated with the promotion of hematoma absorption. The specific neuroprotection induced by TAK-242 was
associated with the inhibition of TLR4 signaling and the upregulation of CD36 expression.
Phagocytosis is essential for hematoma absorption. However, the
injury to perihematomal cells caused by inflammatory factors and
oxygen-free radicals released during the phagocytosis of RBCs
requires further attention (2). Our results show that TAK-242
promoted phagocytosis, increased catalase expression, and reduced H2O2 production. Therefore, the promotion of hematoma
absorption by TAK-242 did not result in a concomitant increase in
the production of free radicals or damage to perihematomal tissues. This finding confirms that TAK-242 represents a potentially
viable treatment option for patients with ICH. Because TLR4
plays an important role in the immune response, complete inhibition of TLR4 would not be appropriate for all scenarios. Our
research moderately regulated TLR4 to restrain excessive inflammation; subsequent research regarding the regulation of the
TLR4 signaling pathway is under way.
The authors have no financial conflicts of interest.
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Disclosures
CD36-MEDIATED HEMATOMA ABSORPTION